CN116478949A - Lignan oxymethyl transferase and use thereof - Google Patents
Lignan oxymethyl transferase and use thereof Download PDFInfo
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- CN116478949A CN116478949A CN202310325177.8A CN202310325177A CN116478949A CN 116478949 A CN116478949 A CN 116478949A CN 202310325177 A CN202310325177 A CN 202310325177A CN 116478949 A CN116478949 A CN 116478949A
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- polypeptide molecule
- lignan
- molecule
- gomisin
- lignans
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- 238000012546 transfer Methods 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
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- 229940118696 vibrio cholerae Drugs 0.000 description 1
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Abstract
The present invention relates to a polypeptide molecule having an amino acid sequence selected from the group consisting of: (A) SEQ ID NO.2; (B) An amino acid sequence which is generated by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and (C) a truncate of (A) or (B); wherein the polypeptide molecule has an activity of catalyzing methylation modification of lignans. The lignanoid oxymethyl transferase can methylate the dibenzylbutane lignanoid and the diphenyl cyclooctene lignanoid, can be used for exploring the lignanoid with novel functions, lays a foundation for the research of the synthesis biology of the lignanoid diversification in the schisandra chinensis, and has great application value. The compound obtained by using the lignan oxymethyl transferase or the compound obtained by using the lignan oxymethyl transferase as an intermediate can be used as an active ingredient of medicines, functional food materials and the like, so that the lignan oxymethyl transferase has positive application prospect in the medicine industry and the food industry.
Description
Technical Field
The invention relates to the technical fields of genetic engineering and enzyme engineering, in particular to an oxymethyl transferase participating in biosynthesis of schisandra dibenzocyclooctene lignans and precursor dibenzylbutane lignans thereof and application thereof.
Background
Lignans are a major class of secondary metabolites in nature, and have been isolated from a variety of medicinal and edible plants, with a variety of structures and new structures being discovered in recent years. Pharmacological activity also covers a variety of aspects including anti-inflammatory, antioxidant, antiviral, neuroprotective, and the like. The lignans with various structures form the medicinal active foundation of the schisandra chinensis.
A variety of lignans from Schisandra chinensis have been isolated, and dibenzocyclooctene lignans and bisbenzylbutane lignans are the main types of lignans in Schisandra chinensis (Schisandra chinensis). Wherein the dibenzylbutane lignans include Pregomisin (Pregomisin, CAS number 66280-26-0), etc.; the dibenzocyclooctene lignans include Gomisin J (Gomisin J, CAS number 66280-25-9), and the like.
The chemical structure research of lignans shows that the structural modification of lignans can improve the physical and chemical properties of drugs, which is beneficial to the interaction between the drugs and receptors or enzymes, and causes corresponding biochemical and biophysical reactions.
Therefore, many researchers often select natural lignans as lead compounds, and derivatize the structures of the natural lignans by adopting chemical methods such as esterification, neutralization, substitution, acylation and the like so as to improve the solubility of the compounds, expand the clinical application range of the compounds and develop a large number of lignans derivatives with novel structures. For example, kuo et al performed an acylation reaction of the hydroxyl group at position 14 of (+) -gomisin K3, and reacted with various acid chlorides and sulfonyl chlorides, respectively, to synthesize 8 new compounds. Pharmacological and toxicological studies show that the derivatives can be used for treating hepatitis B. The 7 compounds have strong anti-hepatitis B surface antigen (HBsAg) effect, and the toxicity of the compound is obviously reduced compared with (+) -gomisin K3. Liu et al modified schisandra chinensis esters by acylation reaction to obtain 10 new derivatives, and screening results of in vitro antitumor effect show that two compounds have cytotoxicity on human glioma U87 cells.
However, lignans are far more than this, and studies on structural modification of lignans are far more than this. More novel lignan structure modification and modification methods are urgently needed to be researched.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a polypeptide molecule, the amino acid sequence of which is selected from the group consisting of: (A) SEQ ID NO.2; (B) An amino acid sequence which is generated by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and (C) a truncate of (A) or (B); wherein the polypeptide molecule has an activity of catalyzing methylation modification of lignans, and further, the polypeptide molecule has an activity of catalyzing methylation of phenolic hydroxyl groups of lignans.
A polypeptide molecule as described above, wherein said lignan is a dibenzylbutane-type lignan or a bisbicyclo octene-type lignan.
The polypeptide molecule as described above, wherein the lignan is regoramin (Pregomisin) or regoramin J (Gomisin J).
A polynucleotide molecule encoding a polypeptide molecule according to any one of the preceding claims, said polynucleotide molecule being selected from the group consisting of: (a) SEQ ID NO.1; (b) A nucleic acid sequence having more than 80% homology with SEQ ID NO.1; and (c) a truncate of (a) or (b).
A recombinant vector comprising: a polynucleotide molecule as described above; an expression vector.
A fused cell comprising: a recombinant vector as described above; and expressing the cells.
A method of preparing a protein for catalyzing methylation modification of lignans, comprising: transforming the nucleic acid molecule of 4 above into an expression cell; a protein expressing the polypeptide molecule in an expression cell; and purifying the protein of the polypeptide molecule.
Use of a polypeptide molecule as described in any one of the preceding claims for catalyzing methylation modification of lignans.
A process for preparing Gomisin K2 (Gomisin K2) and Gomisin K2 (Gomisin K2) prepared by the process, the process comprising: gomisin J (Gomisin J) is catalyzed with one or more selected from the group: a polypeptide molecule as claimed in any one of the preceding claims; a polypeptide molecule encoded by a nucleic acid molecule as described above; a polypeptide molecule expressed by a recombinant vector as described above; a polypeptide molecule obtained by fusing cells as described above; and polypeptide molecules prepared by the methods described above.
A method for preparing a schisandra lignan compound D (Schisandrathera D) and a schisandra lignan compound D (Schisandrathera D) prepared by the method, the method comprising: pre-gomisin (Pregomisin) is catalyzed with one or more of the following groups: a polypeptide molecule according to any one of the preceding claims; a polypeptide molecule encoded by a nucleic acid molecule as described above; a polypeptide molecule expressed by a recombinant vector as described above; a polypeptide molecule obtained by fusing cells as described above; and polypeptide molecules prepared by the methods described above.
The lignanoid oxymethyl transferase can methylate the dibenzylbutane lignanoid and the diphenyl cyclooctene lignanoid, can be used for exploring the lignanoid with novel functions, lays a foundation for the research of the synthesis biology of the lignanoid diversification in the schisandra chinensis, and has great application value. The compound obtained by using the lignan oxymethyl transferase or the compound obtained by using the lignan oxymethyl transferase as an intermediate can be used as an active ingredient of medicines, functional food materials and the like, so that the lignan oxymethyl transferase has positive application prospect in the medicine industry and the food industry.
Drawings
Preferred embodiments of the present invention will be described in further detail below with reference to the attached drawing figures, wherein:
FIG. 1 is an SDS-PAGE electrophoresis of SchOMT2 protein according to an embodiment of the invention, wherein: m: protein molecular mass standard; lane 1: supernatant of SchOMT 2; lane 2: schOMT2 precipitated cells; lane 3: purified protein of SchOMT 2;
FIG. 2 is an HPLC chart of an enzyme-catalyzed reaction of SchOMT2 with pre-gomisin J of Mi Xinji as substrate in accordance with one embodiment of the present invention; wherein, fig. 2a is an HPLC profile of an enzymatic activity reaction of SchOMT2 with gomisin J as substrate according to an embodiment of the invention; FIG. 2b is an HPLC chart of an enzymatic reaction of SchOMT2 pre-gomisin as substrate according to one embodiment of the invention; each enzyme activity catalytic reaction takes the catalytic reaction of the empty carrier as a control;
FIG. 3 is a LC-MS spectrum and reaction formula of an enzyme-catalyzed reaction of SchOMT2 with gomisin J as substrate according to one embodiment of the invention; and
FIG. 4 is a LC-MS spectrum and reaction scheme of an enzyme-catalyzed reaction of SchOMT2 pre-gomisin as substrate according to one embodiment of the invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof, and in which is shown by way of illustration specific embodiments in which the application may be practiced. In the drawings, like reference numerals describe substantially similar components throughout the different views. Various specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to practice the teachings of the present application. It is to be understood that other embodiments may be utilized or structural, logical, or electrical changes may be made to the embodiments of the present application.
The terms described herein have the following meanings:
the term "lignans" as used herein refers to a class of natural organic compounds polymerized in different forms from 2 or 3 molecule phenylpropanoid derivatives, which are distributed throughout angiosperms and gymnosperms. Two main categories can be distinguished: a compound polymerized from two molecules of phenylpropyl derivative through its side chain beta-position is called lignan; compounds in which one molecule of phenylpropyl side chains is linked to the benzene ring of another molecule, or two moieties are linked by an oxygen atom, are called neolignans. More than 200 lignans and more than 100 neolignans have been isolated from the plant kingdom. Chinese medicinal materials of schisandra fruit, acanthopanax root, cuisine, arctium fruit, polygala root, capsule of weeping forsythia, carthamus flower and asarum herb, etc. all contain lignans. Many lignan components may have stereoisomers due to saturated cyclic moieties. Various lignans have been found to have activities of anticancer, antifungal, insecticidal, leukocyte-increasing, glutamic pyruvic transaminase-reducing, liver-protecting, cough-relieving, purgation, etc., but are not so much used as pharmaceutical agents. The derivative of podophyllotoxin is used as anticancer agent, and lignanoid in fructus Schisandrae chinensis has effect in reducing glutamic pyruvic transaminase, and can be used for treating hepatitis.
As used herein, "Schisandra chinensis" refers to Schisandra chinensis, a woody plant of the genus Schisandra of the family Schisandraceae. Researches show that the schisandra chinensis has wide medicinal value, including: the composition has the effects of resisting liver injury, widely inhibiting central nervous system, strengthening heart, enhancing body defensive ability against nonspecific stimulation, inhibiting bacillus anthracis, staphylococcus aureus, staphylococcus albus, typhoid bacillus, vibrio cholerae and the like. Wherein the lignans are main effective components of fructus Schisandrae chinensis, the total lignans content in fructus Schisandrae chinensis fruit is 18.1%, and the total lignans content in stem is 10.25%. The lignan component has obvious antioxidation effect, and can protect heart from lipid peroxidation injury.
As used herein, "methylation" refers to the process of catalytically transferring methyl groups from an active methyl compound to other compounds, which may form various methyl compounds, or which may chemically modify certain proteins or nucleic acids, etc., to form a methylated product. In biological systems, methylation is enzymatically catalyzed, and involves regulation of gene expression, regulation of protein function, and ribonucleic acid processing.
As used herein, "OMT2" belongs to the OMTs family and is an oxygen methyl transferase (O-methyl transferases). The oxymethyl transferase (OMT) is an important enzyme which depends on S-adenosylmethionine to catalyze the generation of various secondary metabolites such as flavonoid, alkaloid, plant protection agent and the like, and plays an important role in various stages of plant growth and development, resisting invasion of external bacteria and the like. OMT is usually present in the animal and plant in the form of a gene family.
The protein of the present invention, for example, the amino acid sequence shown in SEQ ID No.2, comprises (A) a protein having the amino acid sequence shown in SEQ ID No.2, or (B) a protein having an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the above amino acid sequence and having a phenolic hydroxyl methylation function of primisin and/or primin J, or (C) a truncated form of the above two, such as a truncated N-terminal or C-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids, or a protein having an extended N-terminal or C-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids and having a phenolic hydroxyl methylation function of primin J.
As used herein, "substitution, deletion, or addition of one or more bases" or "substitution, deletion, or addition of one or more amino acids" refers to the use of well-known mutagenesis methods such as site-directed mutagenesis [ Hashimoto-Gotoh, gene 152, 271-275 (1995) others ] to mutate a nucleotide sequence or amino acid sequence to a sequence having at least 80% homology to the original sequence. For example, the mutated sequence has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology to the original sequence. In some embodiments, the term "95% or more homology" in the above proteins and genes may be at least 96%, 97%, 98% identical. The term "homology above 90% may be at least 91%, 92%, 93%, 94% identical. The term "above 85% homology" may be at least 86%, 87%, 88%, 89% identical. The term "80% homology or greater" may be at least 81%, 82%, 83%, 84% identical.
The terms "coding gene", "nucleic acid molecule" refer to a Ribonucleotide (RNA) or Deoxyribonucleotide (DNA) sequence that can encode a peptide chain of a particular amino acid. In some embodiments, the encoding gene or nucleic acid molecule may be obtained by total gene synthesis, PCR amplification, chemical synthesis, or the like. The present application is not limited to the manner in which the encoding genes and nucleic acid molecules are obtained.
The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.
In some embodiments of the invention, the term "recombinant vector" is specifically a recombinant vector obtained by cloning a gene encoding a lignan oxymethyl transferase between expression vectors, such as pET28a vectors, which contain cleavage sites, such as bamhi and Xho i. In other embodiments, the expression vector is selected from one or more of the following vectors: other vectors such as pPIC9, pPIC9K, pPICZ a B, pPICZ a B vector, pET series vector, pGEX series vector, pMAL series vector, pQE series vector, pBADmycHis series vector, pTrcHis series vector, pTXB series vector, T series vector, and the like, and modified vectors of the above vectors. In some embodiments, the recombinant expression vector may be selected from prokaryotic expression vectors such as pET series, pGEX series, pCold series, etc., yeast expression vectors such as pPIC9, pHIL-D2, pPIC3.5, etc., and plant expression vectors such as PBI series, pCAMBIA series, etc. The present application is not limited to the manner in which the recombinant vector is obtained, and recombinant vectors of the present application obtained by other means are also within the scope of the present application.
The terms "recombinant expression cell", "recombinant cell", "expression cell" refer to a cell having an exogenous gene integrated within its genome, or a host cell comprising an expression vector in vivo. In some embodiments, the "recombinant expression cell", "recombinant cell", "expression cell" may be a bacterial, fungal, higher plant cell, or the like. In some embodiments, the gene encoding a lignan oxymethyl transferase is obtained by introducing the gene into the E.coli, mammalian cells, yeast cells or Pichia pastoris genetically engineered with glycosylation modification pathways.
The term "comprising" as used herein generally refers to the meaning of containing, summarizing, comprising, or including. In some cases, the meaning of "as", "consisting of … …" is also indicated.
The oxygen methyl transferase (O-methyl transferases) is taken as an important structure modification enzyme and is a key enzyme for biosynthesis of Chinese medicine active compounds in schisandra chinensis. Aiming at the research on the biosynthesis path of the schisandra chinensis dibenzocyclooctene lignans, the path is deduced to be that the dibenzo-benzyl butane lignans are connected through benzene rings to form the dibenzocyclooctene lignans structure. Methylation modification may occur before or after coupling and is an important ring in the synthetic pathway of compounds containing oxymethyl groups.
Therefore, the invention focuses on screening and identifying structural modification genes OMTs of schisandra lignans. The research result has important theoretical research significance and practical application significance for clarifying lignan synthesis pathway of schisandra chinensis, formation mechanism of compound structural diversity and the like.
The invention aims to provide an oxymethyl transferase gene and a protein coded by the same, which can participate in methylation modification of schisandra dibenzocyclooctene lignans and a precursor dibenzylbutane lignans thereof. According to research, the obtained oxymethyl transferase SchOMT2 from schisandra chinensis can catalyze methylation of dibenzocyclooctene type lignans gomisin J and dibenzylbutane type lignans regressine.
The nucleotide sequence of the SchOMT2 gene provided by the invention is shown as SEQ ID No.1 or a mutant sequence thereof.
The amino acid sequence of the protein coded by the SchOMT2 gene is shown as SEQ ID No.2 or a mutant sequence thereof.
The invention can be realized by the following technical scheme:
the technical scheme is as follows: the method for screening the schisandra lignan oxymethyl transferase gene based on transcriptome comprises the following steps:
1) UPLC detects the difference of lignans containing oxymethyl in fruit, mature stem, old leaf and root of schisandra fruit.
2) Identifying members of the gene family of the schisandra chinensis oxymethyl transferase OMTs based on the schisandra chinensis genome, and primarily screening key enzyme protein sequences of the oxymethyl transferase OMTs in the schisandra chinensis by utilizing an HMM model of the OMT gene family.
3) Based on transcriptome data of different tissue parts of the schisandra chinensis, differential gene expression is analyzed, and key enzyme genes for modifying hydroxy methylation in the biosynthesis pathway of the schisandra chinensis lignan are further screened.
The second technical scheme is as follows: functional verification of the key enzyme gene SchOMT 2. The functions of the oxymethyl transferase SchOMT2 were identified in vitro using a prokaryotic expression system with pre-gomisin and gomisin J as substrates.
The present application relates to a polypeptide molecule having an amino acid sequence selected from the group consisting of: (A) SEQ ID NO.2; (B) An amino acid sequence which is generated by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and (C) a truncate of (A) or (B); wherein the polypeptide molecule has an activity of catalyzing methylation modification of lignans, and further, the polypeptide molecule has an activity of catalyzing methylation of phenolic hydroxyl groups of lignans. Further, the above polypeptide molecule is encoded by a coding gene selected from the group consisting of: (a) SEQ ID NO.1; (b) A nucleic acid sequence having more than 80% homology with SEQ ID NO.1; and (c) a truncate of (a) or (b).
In some embodiments, the lignan is a dibenzylbutane lignan or a dibenzocyclooctene lignan. Further, in some embodiments, the dibenzylbutane-type lignan is regoramin (Pregomisin), and the biscyclooctene-type lignan is regoramin J (Gomisin J).
In some embodiments, the present application relates to a recombinant vector comprising: the polynucleotide molecule as described above, which can express the polypeptide molecule as described above. In some embodiments, the polypeptide molecule has activity in catalyzing methylation modification of lignans; an expression vector. In some embodiments, the polypeptide molecule has activity in catalyzing the methylation of lignans phenolic hydroxyl groups. In some embodiments, the expression vector may be selected from prokaryotic expression vectors such as pET series, pGEX series, pCold series, etc., yeast expression vectors such as pPIC9, pHIL-D2, pPIC3.5, etc., and plant expression vectors such as PBI series, pCAMBIA series, etc.
In some embodiments, the present application relates to a fusion cell comprising the aforementioned recombinant vector; and expressing the cells. In some embodiments, the expression cell may be a bacterium, a fungus, a higher plant cell, or the like. In some embodiments, the gene encoding a lignan oxymethyl transferase is obtained by introducing the gene into the E.coli, mammalian cells, yeast cells or Pichia pastoris genetically engineered with glycosylation modification pathways.
In some embodiments, the foregoing polypeptide molecules, or polynucleotide molecules encoding the foregoing polypeptide molecules, may be used to catalyze lignan methylation modifications. In some embodiments, the lignans are dibenzylbutane lignans and/or diphenyl cyclooctene lignans. Further, the dibenzylbutane type lignan is regoramin (Pregomisin), and the biscyclooctene type lignan is regoramin J (Gomisin J). In some embodiments, pregomisin (Pregomisin) may obtain schisandra lignan compound D (Schisandrathera D) under the oxymethyl catalytic activity of the aforementioned polypeptide molecules; in some embodiments, gomisin J (Gomisin J) is capable of obtaining Gomisin K2 (Gomisin K2) under the oxymethyl catalytic activity of the aforementioned polypeptide molecules.
In some embodiments, the schisandra lignan compound D (Schisandrathera D) has the following structural formula:
in some embodiments, gomisin K2 (Gomisin K2) has the following structural formula:
the invention discloses a screening and functional identification method of an oxymethyl transferase coding gene SchOMT2 for catalyzing hydroxy methylation in a lignan biosynthesis pathway in schisandra chinensis, which verifies that SchOMT2 has the function of catalyzing gomisin J to generate gomisin K2, can catalyze hydroxy methylation of pre-gomisin to generate a schisandra chinensis lignan compound D (Schisandrathera D), lays a foundation for the research of synthesis biology of lignan diversification in schisandra chinensis, and has great application value.
The technical solutions of the present application will be explained herein by the following examples.
EXAMPLE 1 cloning of expressed Gene SchOMT2
1.1 extraction of Schisandra chinensis total RNA by CTAB-PVP method
(1) Fresh schisandra plant material (leaf or fruit) is taken and rapidly ground into powder in liquid nitrogen.
(2) Estimating 50-100mg of powder by using a centrifuge tube, adding 600-800 mu L of CTAB-PVP extracting solution preheated at 65 ℃ into a 2mL inlet centrifuge tube precooled in advance, and placing the mixture in a vortex oscillator to oscillate for 30s to enable the mixture to be fully cracked.
The preparation method of the CTAB-PVP extraction buffer solution comprises the following steps:
100mM Tris-HCl (pH 8.0), 2% CTAB (w/v), 2% PVP (w/v), 25mM EDTA,2M NaCl, mercaptoethanol added to 0.2% after autoclaving; solution configuration double distilled water treated with DEPC (ddH 2 O), autoclaving and then spare.
(3) The mixture was mixed by inverting it every 10 minutes in a water bath at 65℃for 30 minutes.
(4) After cooling to room temperature, 600-800. Mu.L of chloroform was added, and after mixing was reversed, centrifugation was carried out at 13,000rpm for 10min at 4 ℃.
(5) Transferring the supernatant to a new centrifuge tube with 2mL inlet, adding 600-800 μl of chloroform, shaking, mixing well, and centrifuging at 4deg.C at 13,000rpm for 10min.
(6) The above procedure was repeated (i.e., three extractions with chloroform).
(7) The supernatant was carefully pipetted into a fresh imported 1.5mL centrifuge tube, 1/3 of the 8M LiCl was added and allowed to stand at-20℃overnight.
(8) Centrifuge at 13,000rpm for 10min at 4℃and discard the supernatant.
(9) The precipitate was washed 2-3 times with 700. Mu.L of 75% ethanol (DEPC water formulation). Centrifuging, discarding the supernatant, and volatilizing residual ethanol.
(10) The total RNA was prepared by dissolving RNA in 30. Mu.L of sterilized water treated with protease K (Proteinase K). The concentration and quality of the extracted RNA were measured using a nucleic acid protein meter model BioPhotometer plus.
1.2SchOMT2 Gene full-Length amplification
1.2.1 primer design
The full-length primer SchOMT2-F/R was designed on both sides of the Open Reading Frame (ORF) using the software SnapGene to amplify the gene.
1.2.2cDNA Synthesis
The cDNA template strand is obtained by PCR technology with the total RNA of the extracted schisandra chinensis as a template and a PrimerScript RT Master Mix reverse transcription system.
The reverse transcription system and reverse transcription procedure were as follows:
reverse transcription PCR system:
reverse transcription procedure: 37 ℃ for 15min;
85℃,15s。
the reverse transcription product was stored at-20℃and diluted before use.
1.2.3 amplification of the Gene of interest
The diluted reverse transcribed Schisandra chinensis cDNA is used as a template and SchOMT2-F/R is used as a primer for amplification.
The amplification system and the amplification procedure were as follows:
the components are added into a 200 mu L PCR tube to be uniformly mixed, and the mixture is put into a PCR instrument for amplification after low-speed centrifugation according to the following procedures:
95℃,3min；
95 ℃ for 15s;52 ℃,15s;72 ℃,45s,30 cycles;
72℃,5min。
and (3) detecting the PCR reaction product by agarose gel electrophoresis, wherein the electrophoresis result is shown in figure 1, and cutting the target size strip into gel and recovering.
Example 2 construction of expression vector for SchOMT2 Gene
2.1 amplification of SchOMT2 fragment with homology arms
The SchOMT2 fragment was amplified with the homology arm primer SchOMT2-pET28 a-F/R. The amplification system and procedure are shown in example 1.
2.2 construction of expression vectors by homologous recombination
2.2.1 vector double enzyme digestion
Vector pET28a was digested with BamH I and Xho I in the following manner:
the enzyme digestion is carried out in a water bath at 37 ℃ for 30min. And adding 10 Xsample buffer (Loading buffer) into the enzyme digestion product to stop the reaction, then carrying out agarose gel electrophoresis, and selecting a proper strip for gel recovery, wherein the gel recovery method is the same as that described above and is not repeated.
2.2.2 homologous recombination, transformation and Positive validation
The target fragment with the homology arm is connected with the vector pET28a after enzyme digestion by a homologous recombination kit, and the system is as follows:
the components are fully and evenly mixed, reacted for 30min at 37 ℃, and immediately placed on ice for cooling. The ligation product was competent to transform E.coli DH 5. Alpha. Taking out competent cells of Escherichia coli DH5 alpha stored at-80 ℃, thawing on ice, adding all the connection products, gently blowing and mixing, and standing on ice for 30min; after 45s of heat shock in a water bath at 42 ℃, the mixture is rapidly placed on ice for 2min, 500 mu L of antibiotic-free LB culture medium is added, then the mixture is subjected to shaking culture for 1h in a culture box at 37 ℃, 200 mu L of transformation liquid is coated on LB solid culture medium (containing 100 mu g/mL kanamycin resistance), and the mixture is subjected to static culture at 37 ℃ for 12h to 16h.
And selecting the monoclonal to perform colony PCR (polymerase chain reaction) to verify positive, amplifying a bright monoclonal with single target size band as positive, sequencing the positive clone, taking the correctly sequenced monoclonal to store bacteria, and extracting the SchOMT2-pET32a plasmid. The constructed prokaryotic expression vector plasmid is transformed into competent cells of escherichia coli BL21 (DE 3) by a thermal shock method, and the transformation, screening and identification methods are the same as those described above and are not repeated.
Example 3 Gene protein expression and enzymatic Activity function analysis
3.1 prokaryotic expression of SchOMT2 recombinant protein
(1) The strain SchOMT2-pET28a-BL21 positive clone was picked and inoculated into 4mL of LB medium containing kanamycin (Kana) resistance, and shake cultured overnight at 37℃and 110 rpm.
(2) Inoculating the cultured bacterial liquid into 200mL of Kana-resistant culture medium according to the ratio of 1:100, and culturing under the same condition until OD 600 And approximately 0.5. Adding 0.3mM IPTG into the bacterial liquid, and culturing in a shaking table at 16 ℃ and 110rpm for 16-18 hours to induce the expression of the target protein.
(3) And (3) bacterial collection: the bacterial solution was centrifuged at 5000rpm and the supernatant was discarded after 5 minutes.
(4) Washing: adding a proper amount of Binding buffer into a centrifuge tube according to the bacterial amount, re-suspending the bacterial body, centrifuging at 5,000rpm for 5min, collecting bacterial body, washing twice, and adding 15-20 mL of Binding buffer to re-suspend the bacterial body.
(5) Cracking: placing the bacterial liquid into an ice-water mixture, performing ultrasonic bacterial lysis on the bacterial liquid, centrifuging at 12,000rpm at 4 ℃ for 20min, and collecting the supernatant to obtain crude enzyme.
3.2 functional verification of SchOMT2 crude enzyme
The SchOMT2 performs in vitro enzyme activity function identification, and takes a reaction system added with pET28a protein as a control group. The substrate is pre-gomisin, gomisin J and gomisin K1. The enzyme activity reaction system is as follows:
the above components were mixed and allowed to react overnight at 30℃and then quenched by adding 50. Mu.L of methanol, and the supernatant was centrifuged at 12,000rpm for 30 minutes and analyzed by HPLC for enzymatic activity.
The results are shown in figure 2, where SchOMT2 catalyzes the methylation of the dibenzocyclooctene lignans gomisin J and the dibenzylbutane lignans pregamicin compared to the blank.
Purification and functional verification of 3.3pchomt 2 recombinant protein
Expression of recombinant proteins As described in 3.1, the supernatant of the disrupted bacterial solution was subjected to column purification, and a portion of the supernatant was prepared for SDS-PAGE to observe the protein expression.
(1) Separating: the collected supernatant was fed to an equilibrated Ni-NTA column, and after the supernatant was drained, a column volume of eluent (containing 20mM imidazole) was added to wash out the impurity proteins, followed by collection of the recombinant protein of interest with 5mL of Elution buffer (Elution buffer) (containing 250mM imidazole).
(2) Ultrafiltration: placing the eluted protein solution into a ultrafiltration tube with a protein molecule of 30,000Da, centrifuging for 10min at 4,000rcf, adding Binding buffer (Binding buffer) for 2-3 times, and concentrating target protein.
(3) The concentrate was aspirated into a 2mL collection tube, the protein concentration was determined, and the sample was left.
(4) After adding 10% glycerol into the protein, quick freezing the protein with liquid nitrogen and storing the protein in a refrigerator at the temperature of minus 80 ℃ for standby.
Binding buffer (Binding buffer): 2.42g Tris-HCl and 29.22g NaCl are respectively weighed, dissolved in water, pH is regulated to 8.0, volume is fixed to 1000mL, 70 mu L beta-mercaptoethanol is added after sterilization, and the mixture is preserved at 4 ℃.
Elution buffer (Elution buffer): 2.42g Tris-HCl, 29.22g NaCl and 34gimidazole are respectively weighed, dissolved in water, pH is regulated to 8.0, the volume is fixed to 1000mL, 70 mu L beta-mercaptoethanol is added after sterilization, and the mixture is preserved at 4 ℃.
Activity verification of purified proteins enzyme activity assay was performed using LC-MS as described in 3.2. The results are shown in fig. 3 and 4. Wherein, the molecular weight of the products produced by the SchOMT2 catalyzed gomisin J shown in FIG. 3 and the SchOMT2 catalyzed pre-gomisin shown in FIG. 4 are both the molecular weight added with one methyl group.
The above embodiments are provided for illustrating the present invention and not for limiting the present invention, and various changes and modifications may be made by one skilled in the relevant art without departing from the scope of the present invention, therefore, all equivalent technical solutions shall fall within the scope of the present disclosure.
Claims (10)
1. A polypeptide molecule having an amino acid sequence selected from the group consisting of:
(A)SEQ ID NO.2；
(B) An amino acid sequence which is generated by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and
(C) A truncate of (a) or (B);
wherein the polypeptide molecule has an activity of catalyzing the methylation of the phenolic hydroxyl group of lignans.
2. The polypeptide molecule of claim 1, wherein the lignan is a dibenzylbutane-type lignan or a dibenzocyclooctene-type lignan.
3. The polypeptide molecule of claim 2, wherein the lignan is regoramin (Pregomisin) or regoramin J (Gomisin J).
4. A polynucleotide molecule encoding the polypeptide molecule of any one of claims 1-3, said polynucleotide molecule selected from the group consisting of:
(a)SEQ ID NO.1；
(b) A nucleic acid sequence having more than 80% homology with SEQ ID NO.1; and
(c) The truncate of (a) or (b).
5. A recombinant vector comprising:
the polynucleotide molecule of claim 4; and
an expression vector.
6. A fused cell comprising:
the recombinant vector of claim 5; and
and expressing the cells.
7. A method of preparing a protein for catalyzing methylation modification of lignans, comprising:
transforming the nucleic acid molecule of claim 4 into an expression cell;
a protein expressing the polypeptide molecule in an expression cell; and
purifying the protein of the polypeptide molecule.
8. Use of a polypeptide molecule according to any one of claims 1-3 for catalyzing methylation modification of lignans.
9. A process for the preparation of Gomisin K2 (Gomisin K2), the process comprising: gomisin J (Gomisin J) is catalyzed with one or more selected from the group:
a polypeptide molecule according to any one of claims 1 to 3;
a polypeptide molecule encoded by the nucleic acid molecule of claim 4;
a polypeptide molecule expressed by the recombinant vector of claim 5;
a polypeptide molecule obtained from the fusion cell of claim 6; and
a polypeptide molecule prepared by the method of claim 7.
10. A method of preparing a schisandra lignan compound D (Schisandrathera D), the method comprising: pre-gomisin (Pregomisin) is catalyzed with one or more of the following groups:
a polypeptide molecule according to any one of claims 1 to 3;
a polypeptide molecule encoded by the nucleic acid molecule of claim 4;
a polypeptide molecule expressed by the recombinant vector of claim 5;
a polypeptide molecule obtained from the fusion cell of claim 6; and
a polypeptide molecule prepared by the method of claim 7.
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